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- 详细信息
- 文献和实验
- 技术资料
- 保存条件：
-20
- 保质期：
2年
- 英文名：
pGWB17
- 库存：
100
- 供应商：
上海烜雅生物科技有限公司
- 规格：
干粉/液体
基本信息
名称：pGWB17植物表达质粒
别称: pGWB17
质粒属性
质粒简介
pGWB17是一个GateWay系列的植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17407 bp ds-DNA circular SYN 27-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..17407
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/label=TetR
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
promoter 2634..2817
/function="NOS promoter"
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 5461..5806
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
protein_bind 5828..5952
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 5989..6019
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 6073..6732
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 7074..7379
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(7420..7544)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 7570..7599
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7606..7635
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7651..7680
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7687..7716
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
terminator 7747..7999
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8428..8680
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(9148..10173)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(10183..10527)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 11271..11295
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12891..14039)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(14338..15132)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15324..16091
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16714..18
/label=oriV
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 gttatcaaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
5881 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
5941 ccagtcacta tggcggcccc tcgaggggcc gcattaggca ccccaggctt tacactttat
6001 gcttccggct cgtataatgt gtggattttg agttaggatc cggcgagatt ttcaggagct
6061 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
6121 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
6181 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
6241 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca
6301 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
6361 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
6421 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
6481 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
6541 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
6601 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtctgt
6661 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
6721 ggcggggcgt aaagatctgg atccggctta ctaaaagcca gataacagta tgcgtatttg
6781 cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa
6841 agaggtgtgc tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc
6901 tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc
6961 ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc
7021 ccggtttatt gaaatgaacg gctcttttgc tgacgagaac agggactggt gaaatgcagt
7081 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
7141 atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
7201 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
7261 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
7321 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
7381 tgtcaggctc ccttatacac agccagtctg caggtcgacc atagtgactg gatatgttgt
7441 gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt
7501 atatcatttt acgtttctcg ttcagctttc ttgtacaaag tggttgataa cagcgggtta
7561 attaacggtg aacaaaagct aatctccgag gaagacttga acggtgaaca aaaattaatc
7621 tcagaagaag acttgaacgg actcgacggt gaacaaaagt tgatttctga agaagatttg
7681 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtagcgctta gagctcgaat
7741 ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt
7801 cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg
7861 taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt
7921 taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg
7981 tcatctatgt tactagatcg ggaattagct tcatcaacgc aagacatgcg cacgaccgtc
8041 tgacaggaga ggaatttccg acgagcacag aaaggacttg ctcttggacg taggcctatt
8101 tctcaggcac atgtatcaag tgttcggacg tgggttttcg atggtgtatc agccgccgcc
8161 aactgggaga tgaggaggct ttcttggggg gcagtcagca gttcatttca caagacagag
8221 gaacttgtaa ggagatgcac tgatttatct tggcgcaaac cagcaggacg aattagtggg
8281 aatagcccgc gaatatctaa gttatgcctg tcggcatgag cagaaacttc caattcgaaa
8341 cagtttggag aggttgtttt tgggcatacc ttttgttagt cagcctctcg attgctcatc
8401 gtcattacac agtaccgaag tttgatcgat ctagtaacat agatgacacc gcgcgcgata
8461 atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg
8521 ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta
8581 catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat
8641 tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc tgcttcgacg cactccttct
8701 ttactccacc atctcgtcct tattgaaaac gtgggtagca ccaaaacgaa tcaagtcgct
8761 ggaactgaag ttaccaatca cgctggatga tttgccagtt ggattaatct tgcctttccc
8821 cgcatgaata atattgatga atgcatgcgt gaggggtatt tcgattttgg caatagctgc
8881 aattgccgcg acatcctcca acgagcataa ttcttcagaa aaatagcgat gttccatgtt
8941 gtcagggcat gcatgatgca cgttatgagg tgacggtgct aggcagtatt ccctcaaagt
9001 ttcatagtca gtatcatatt catcattgca ttcctgcaag agagaattga gacgcaatcc
9061 acacgctgcg gcaaccttcc ggcgttcgtg gtctatttgc tcttggacgt tgcaaacgta
9121 agtgttggat cccggtcggc atctactcta ttcctttgcc ctcggacgag tgctggggcg
9181 tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct
9241 gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg
9301 accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc
9361 aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct
9421 ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat
9481 gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt
9541 tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg
9601 gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc
9661 actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca
9721 tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc
9781 gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag
9841 aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga
9901 gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag
9961 cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct
10021 atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc
10081 gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc
10141 gacagacgtc gcggtgagtt caggcttttt catatcgggg tcgtcctctc caaatgaaat
10201 gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt
10261 acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct
10321 ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca
10381 tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt
10441 tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc
10501 gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg
10561 atattcttgg agtagacgag agtgtcgtgc tccaccatgt tgacggatct ctaggacgcg
10621 tcctagaagc taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
10681 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
10741 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgcc cgctcctttc
10801 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
10861 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
10921 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
10981 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
11041 atctcgggct attcttttga tttataaggg attttgccga tttcggaacc accatcaaac
11101 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
11161 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccccag
11221 tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca
11281 caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca
11341 ccactcgata caggcagccc atcagtccgg gacggcgtca gcgggagagc cgttgtaagg
11401 cggcagactt tgctcatgtt accgatgcta ttcggaagaa cggcaactaa gctgccgggt
11461 ttgaaacacg gatgatctcg cggagggtag catgttgatt gtaacgatga cagagcgttg
11521 ctgcctgtga tcaaatatca tctccctcgc agagatccga attatcagcc ttcttattca
11581 tttctcgctt aaccgtgaca ggctgtcgat cttgagaact atgccgacat aataggaaat
11641 cgctggataa agccgctgag gaagctgagt ggcgctattt ctttagaagt gaacgttgac
11701 gatatcaact cccctatcca ttgctcaccg aatggtacag gtcggggacc cgaagttccg
11761 actgtcggcc tgatgcatcc ccggctgatc gaccccagat ctggggctga gaaagcccag
11821 taaggaaaca actgtaggtt cgagtcgcga gatcccccgg aaccaaagga agtaggttaa
11881 acccgctccg atcaggccga gccacgccag gccgagaaca ttggttcctg taggcatcgg
11941 gattggcgga tcaaacacta aagctactgg aacgagcaga agtcctccgg ccgccagttg
12001 ccaggcggta aaggtgagca gaggcacggg aggttgccac ttgcgggtca gcacggttcc
12061 gaacgccatg gaaaccgccc ccgccaggcc cgctgcgacg ccgacaggat ctagcgctgc
12121 gtttggtgtc aacaccaaca gcgccacgcc cgcagttccg caaatagccc ccaggaccgc
12181 catcaatcgt atcgggctac ctagcagagc ggcagagatg aacacgacca tcagcggctg
12241 cacagcgcct accgtcgccg cgaccccgcc cggcaggcgg tagaccgaaa taaacaacaa
12301 gctccagaat agcgaaatat taagtgcgcc gaggatgaag atgcgcatcc accagattcc
12361 cgttggaatc tgtcggacga tcatcacgag caataaaccc gccggcaacg cccgcagcag
12421 cataccggcg acccctcggc ctcgctgttc gggctccacg aaaacgccgg acagatgcgc
12481 cttgtgagcg tccttggggc cgtcctcctg tttgaagacc gacagcccaa tgatctcgcc
12541 gtcgatgtag gcgccgaatg ccacggcatc tcgcaaccgt tcagcgaacg cctccatggg
12601 ctttttctcc tcgtgctcgt aaacggaccc gaacatctct ggagctttct tcagggccga
12661 caatcggatc tcgcggaaat cctgcacgtc ggccgctcca agccgtcgaa tctgagcctt
12721 aatcacaatt gtcaatttta atcctctgtt tatcggcagt tcgtagagcg cgccgtgcgt
12781 cccgagcgat actgagcgaa gcaagtgcgt cgagcagtgc ccgcttgttc ctgaaatgcc
12841 agtaaagcgc tggctgctga acccccagcc ggaactgacc ccacaaggcc ctagcgtttg
12901 caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc tcgcaactct
12961 tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc cgatccgcac
13021 atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct gaaatagtcg
13081 aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa tttcgtgtag
13141 tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca acgggacgtt
13201 ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc caggtgccca
13261 acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca ttcctcggcc
13321 ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc gtagaacgtg
13381 aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg ctgccacacc
13441 agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac gtccttgttg
13501 acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt gcgcgtggtg
13561 aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg ccacggcgca
13621 atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt cagcaacgcg
13681 gcctgcttgg cctcgctgac ctgttttgcc aggtcctcgc cggcggtttt tcgcttcttg
13741 gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc cgcctcctgt
13801 tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg gggagccagt
13861 tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ccatcgagcc gacggactgg
13921 aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc atcctcggcg
13981 gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt ccgattcatt
14041 caccctcctt gcgggattgc cccgactcac gccggggcaa tgtgccctta ttcctgattt
14101 gacccgcctg gtgccttggt gtccagataa tccaccttat cggcaatgaa gtcggtcccg
14161 tagaccgtct ggccgtcctt ctcgtacttg gtattccgaa tcttgccctg cacgaatacc
14221 agcgacccct tgcccaaata cttgccgtgg gcctcggcct gagagccaaa acacttgatg
14281 cggaagaagt cggtgcgctc ctgcttgtcg ccggcatcgt tgcgccacat ctaggtacta
14341 aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct tgatccccag
14401 taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga tcgaccggac
14461 gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat caataaagcc
14521 acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt gggaaaagac
14581 aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa
14641 tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat tcagtaagta
14701 atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg atatgtcgat
14761 ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag ggctttgttc
14821 atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca gattgctcca
14881 gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt ccagccatag
14941 catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc tgtccgtcat
15001 ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag gagacattcc
15061 ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg gtgatattct
15121 cattttagcc atttattatt tccttcctct tttctacagt atttaaagat accccaagaa
15181 gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa ccttaaatac
15241 cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt atcgacggag
15301 ccgattttga aaccacaatt atgggtgatg ctgccaactt actgatttag tgtatgatgg
15361 tgtttttgag gtgctccagt ggcttctgtg tctatcagct gtccctcctg ttcagctact
15421 gacggggtgg tgcgtaacgg caaaagcacc gccggacatc agcgctatct ctgctctcac
15481 tgccgtaaaa catggcaact gcagttcact tacaccgctt ctcaacccgg tacgcaccag
15541 aaaatcattg atatggccat gaatggcgtt ggatgccggg caacagcccg cattatgggc
15601 gttggcctca acacgatttt acgtcactta aaaaactcag gccgcagtcg gtaacctcgc
15661 gcatacagcc gggcagtgac gtcatcgtct gcgcggaaat ggacgaacag tggggctatg
15721 tcggggctaa atcgcgccag cgctggctgt tttacgcgta tgacagtctc cggaagacgg
15781 ttgttgcgca cgtattcggt gaacgcacta tggcgacgct ggggcgtctt atgagcctgc
15841 tgtcaccctt tgacgtggtg atatggatga cggatggctg gccgctgtat gaatcccgcc
15901 tgaagggaaa gctgcacgta atcagcaagc gatatacgca gcgaattgag cggcataacc
15961 tgaatctgag gcagcacctg gcacggctgg gacggaagtc gctgtcgttc tcaaaatcgg
16021 tggagctgca tgacaaagtc atcgggcatt atctgaacat aaaacactat caataagttg
16081 gagtcattac ccaattatga tagaatttac aagctataag gttattgtcc tgggtttcaa
16141 gcattagtcc atgcaagttt ttatgctttg cccattctat agatatattg ataagcgcgc
16201 tgcctatgcc ttgccccctg aaatccttac atacggcgat atcttctata taaaagatat
16261 attatcttat cagtattgtc aatatattca aggcaatctg cctcctcatc ctcttcatcc
16321 tcttcgtctt ggtagctttt taaatatggc gcttcataga gtaattctgt aaaggtccaa
16381 ttctcgtttt catacctcgg tataatctta cctatcacct caaatggttc gctgggttta
16441 tcgcaccccc gaacacgagc acggcacccg cgaccactat gccaagaatg cccaaggtaa
16501 aaattgccgg ccccgccatg aagtccgtga atgccccgac ggccgaagtg aagggcaggc
16561 cgccacccag gccgccgccc tcactgcccg gcacctggtc gctgaatgtc gatgccagca
16621 cctgcggcac gtcaatgctt ccgggcgtcg cgctcgggct gatcgcccat cccgttactg
16681 ccccgatccc ggcaatggca aggactgcca gcgctgccat ttttggggtg aggccgttcg
16741 cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgggc cgggagggtt
16801 cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg cgcacagggc gcagccctgg
16861 ttaaaaacaa ggtttataaa tattggttta aaagcaggtt aaaagacagg ttagcggtgg
16921 ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc tgcctgtgga cagcccctca
16981 aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc cctcaagtgt caaggatcgc
17041 gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat accgcagggc acttatcccc
17101 aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc aggcgttttc gccgatttgc
17161 gaggctggcc agctccacgt cgccggccga aatcgagcct gcccctcatc tgtcaacgcc
17221 gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc ctcatctgtc agtgagggcc
17281 aagttttccg cgaggtatcc acaacgccgg cggccgcggt gtctcgcaca cggcttcgac
17341 ggcgtttctg gcgcgtttgc agggccatag acggccgcca gcccagcggc gagggcaacc
17401 agcccgg
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
名称：pGWB17植物表达质粒
别称: pGWB17
| 启动子: | 35s |
|---|---|
| 复制子: | oriV |
| 原核抗性: | Kan |
| 筛选标记: | Hyg,G418 |
| 克隆菌株: | DB3.1 |
| 培养条件: | 37度 |
质粒属性
| 载体宿主： | 植物 |
|---|---|
| 载体用途： | 蛋白表达 |
| 基因种属： | 空载体 |
| 基因类型： | ORF,GateWay |
| 原核抗性： | Kan |
| 筛选标记： | Neo/G418,Hyg |
| 荧光蛋白： |
质粒简介
pGWB17是一个GateWay系列的植物表达质粒。
质粒图谱
质粒序列
LOCUS Exported 17407 bp ds-DNA circular SYN 27-JAN-2018
DEFINITION synthetic circular DNA
FEATURES Location/Qualifiers
source 1..17407
/organism="synthetic DNA construct"
/mol_type="other DNA"
CDS complement(145..516)
/codon_start=1
/gene="traJ"
/product="oriT-recognizing protein"
/label=traJ
/translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
KQDELGKVMMGVVRPRAEP"
oriT 549..658
/note="incP origin of transfer"
CDS complement(1339..1989)
/codon_start=1
/gene="tetR"
/product="tetracycline resistance regulatory protein"
/label=TetR
/translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
misc_feature 2454..2478
/label=RB T-DNA repeat
/note="right border repeat from nopaline C58 T-DNA"
promoter 2634..2817
/function="NOS promoter"
/label=NOS promoter
/note="nopaline synthase promoter"
CDS 2838..3632
/codon_start=1
/gene="aph(3')-II (or nptII)"
/product="aminoglycoside phosphotransferase from Tn5"
/label=NeoR/KanR
/note="confers resistance to neomycin, kanamycin, and G418
(Geneticin(R))"
/translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
SHLAPAEKVSIMADAMRRLHTLDPAT-CPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
GLAPAELFARLKARMPDGDDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
terminator 4022..4274
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
promoter 5461..5806
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
protein_bind 5828..5952
/gene="mutant version of attR"
/label=attR1
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
promoter 5989..6019
/label=lac UV5 promoter
/note="E. coli lac promoter with an ""up"" mutation"
CDS 6073..6732
/codon_start=1
/gene="cat"
/product="chloramphenicol acetyltransferase"
/label=CmR
/note="confers resistance to chloramphenicol"
/translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
CDS 7074..7379
/codon_start=1
/gene="ccdB"
/product="CcdB, a bacterial toxin that poisons DNA gyrase"
/label=ccdB
/note="Plasmids containing the ccdB gene cannot be
propagated in standard E. coli strains."
/translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLS-DK
VSRELYPVVHIGDESWRMMTTDM-ASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
protein_bind complement(7420..7544)
/gene="mutant version of attR"
/label=attR2
/bound_moiety="LR Clonase(TM)"
/note="recombination site for the Gateway(R) LR reaction"
CDS 7570..7599
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7606..7635
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7651..7680
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
CDS 7687..7716
/codon_start=1
/product="Myc (human c-Myc proto-oncogene) epitope tag"
/label=Myc
/translation="EQKLISEEDL"
terminator 7747..7999
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
terminator 8428..8680
/label=NOS terminator
/note="nopaline synthase terminator and poly(A) signal"
CDS complement(9148..10173)
/codon_start=1
/gene="aph(4)-Ia"
/product="aminoglycoside phosphotransferase from E. coli"
/label=HygR
/note="confers resistance to hygromycin"
/translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
ETELPAVLQPVAEAM-DAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
E"
promoter complement(10183..10527)
/label=CaMV 35S promoter
/note="strong constitutive promoter from cauliflower mosaic
virus"
misc_feature 11271..11295
/label=LB T-DNA repeat
/note="left border repeat from nopaline C58 T-DNA"
CDS complement(12891..14039)
/codon_start=1
/product="trans-acting replication protein that binds to
and activates oriV"
/label=trfA
/translation="MNRTFDRKAYRQELIDAGFSAEDAETIASRTVMRAPRETFQSVGS
MVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
CDS complement(14338..15132)
/codon_start=1
/gene="aphA-3"
/product="aminoglycoside phosphotransferase"
/label=KanR
/note="confers resistance to kanamycin"
/translation="MAKMRISPELKKLIEKYRCVKDTEGMSPAKVYKLVGENENLYLKM
TDSRYKGTTYDVEREKDMMLWLEGKLPVPKVLHFERHDGWSNLLMSEADGVLCSEEYED
EQSPEKIIELYAECIRLFHSIDISDCPYTNSLDSRLAELDYLLNNDLADVDCENWEEDT
PFKDPRELYDFLKTEKPEEELVFSHGDLGDSNIFVKDGKVSGFIDLGRSGRADKWYDIA
FCVRSIREDIGEEQYVELFFDLLGIKPDWEKIKYYILLDELF"
mobile_element 15324..16091
/mobile_element_type="insertion sequence:IS1"
/note="prokaryotic transposable element"
rep_origin 16714..18
/label=oriV
/note="incP origin of replication"
ORIGIN
1 tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc
61 gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc
121 cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat
181 gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc
241 atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag
301 gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc
361 cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc
421 cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg
481 gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc
541 ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata
601 agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg
661 ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata
721 tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta
781 tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg
841 gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt
901 atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag
961 gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
1021 gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta
1081 ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt
1141 cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg
1201 aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga
1261 cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg
1321 ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac
1381 gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc
1441 cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg
1501 ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc
1561 cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc
1621 cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg
1681 cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg
1741 ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg
1801 attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg
1861 ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc
1921 gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa
1981 ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg
2041 ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa
2101 cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc
2161 ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc
2221 gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg
2281 ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc
2341 gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct
2401 tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc
2461 cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga
2521 tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc
2581 gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc
2641 tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc
2701 taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa
2761 attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg
2821 gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
2881 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
2941 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
3001 gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg
3061 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
3121 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
3181 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
3241 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
3301 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
3361 aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga
3421 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
3481 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
3541 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
3601 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
3661 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
3721 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
3781 catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat
3841 cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg
3901 gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg
3961 cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc
4021 cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc
4081 gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg
4141 catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata
4201 cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc
4261 tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg
4321 gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag
4381 gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta
4441 ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca
4501 aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt
4561 ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg
4621 ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt
4681 ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc
4741 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag
4801 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
4861 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
4921 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag
4981 ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg
5041 tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa
5101 ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat
5161 atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc
5221 aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat
5281 ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt
5341 catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca
5401 cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat
5461 tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat
5521 ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg
5581 cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc
5641 cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt
5701 ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca
5761 agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga
5821 gttatcaaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
5881 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
5941 ccagtcacta tggcggcccc tcgaggggcc gcattaggca ccccaggctt tacactttat
6001 gcttccggct cgtataatgt gtggattttg agttaggatc cggcgagatt ttcaggagct
6061 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
6121 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
6181 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
6241 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca
6301 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
6361 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
6421 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
6481 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
6541 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
6601 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtctgt
6661 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
6721 ggcggggcgt aaagatctgg atccggctta ctaaaagcca gataacagta tgcgtatttg
6781 cgcgctgatt tttgcggtat aagaatatat actgatatgt atacccgaag tatgtcaaaa
6841 agaggtgtgc tatgaagcag cgtattacag tgacagttga cagcgacagc tatcagttgc
6901 tcaaggcata tatgatgtca atatctccgg tctggtaagc acaaccatgc agaatgaagc
6961 ccgtcgtctg cgtgccgaac gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc
7021 ccggtttatt gaaatgaacg gctcttttgc tgacgagaac agggactggt gaaatgcagt
7081 ttaaggttta cacctataaa agagagagcc gttatcgtct gtttgtggat gtacagagtg
7141 atattattga cacgcccggg cgacggatgg tgatccccct ggccagtgca cgtctgctgt
7201 cagataaagt ctcccgtgaa ctttacccgg tggtgcatat cggggatgaa agctggcgca
7261 tgatgaccac cgatatggcc agtgtgccgg tctccgttat cggggaagaa gtggctgatc
7321 tcagccaccg cgaaaatgac atcaaaaacg ccattaacct gatgttctgg ggaatataaa
7381 tgtcaggctc ccttatacac agccagtctg caggtcgacc atagtgactg gatatgttgt
7441 gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt
7501 atatcatttt acgtttctcg ttcagctttc ttgtacaaag tggttgataa cagcgggtta
7561 attaacggtg aacaaaagct aatctccgag gaagacttga acggtgaaca aaaattaatc
7621 tcagaagaag acttgaacgg actcgacggt gaacaaaagt tgatttctga agaagatttg
7681 aacggtgaac aaaagctaat ctccgaggaa gacttgaacg gtagcgctta gagctcgaat
7741 ttccccgatc gttcaaacat ttggcaataa agtttcttaa gattgaatcc tgttgccggt
7801 cttgcgatga ttatcatata atttctgttg aattacgtta agcatgtaat aattaacatg
7861 taatgcatga cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt
7921 taatacgcga tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg
7981 tcatctatgt tactagatcg ggaattagct tcatcaacgc aagacatgcg cacgaccgtc
8041 tgacaggaga ggaatttccg acgagcacag aaaggacttg ctcttggacg taggcctatt
8101 tctcaggcac atgtatcaag tgttcggacg tgggttttcg atggtgtatc agccgccgcc
8161 aactgggaga tgaggaggct ttcttggggg gcagtcagca gttcatttca caagacagag
8221 gaacttgtaa ggagatgcac tgatttatct tggcgcaaac cagcaggacg aattagtggg
8281 aatagcccgc gaatatctaa gttatgcctg tcggcatgag cagaaacttc caattcgaaa
8341 cagtttggag aggttgtttt tgggcatacc ttttgttagt cagcctctcg attgctcatc
8401 gtcattacac agtaccgaag tttgatcgat ctagtaacat agatgacacc gcgcgcgata
8461 atttatccta gtttgcgcgc tatattttgt tttctatcgc gtattaaatg tataattgcg
8521 ggactctaat cataaaaacc catctcataa ataacgtcat gcattacatg ttaattatta
8581 catgcttaac gtaattcaac agaaattata tgataatcat cgcaagaccg gcaacaggat
8641 tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc tgcttcgacg cactccttct
8701 ttactccacc atctcgtcct tattgaaaac gtgggtagca ccaaaacgaa tcaagtcgct
8761 ggaactgaag ttaccaatca cgctggatga tttgccagtt ggattaatct tgcctttccc
8821 cgcatgaata atattgatga atgcatgcgt gaggggtatt tcgattttgg caatagctgc
8881 aattgccgcg acatcctcca acgagcataa ttcttcagaa aaatagcgat gttccatgtt
8941 gtcagggcat gcatgatgca cgttatgagg tgacggtgct aggcagtatt ccctcaaagt
9001 ttcatagtca gtatcatatt catcattgca ttcctgcaag agagaattga gacgcaatcc
9061 acacgctgcg gcaaccttcc ggcgttcgtg gtctatttgc tcttggacgt tgcaaacgta
9121 agtgttggat cccggtcggc atctactcta ttcctttgcc ctcggacgag tgctggggcg
9181 tcggtttcca ctatcggcga gtacttctac acagccatcg gtccagacgg ccgcgcttct
9241 gcgggcgatt tgtgtacgcc cgacagtccc ggctccggat cggacgattg cgtcgcatcg
9301 accctgcgcc caagctgcat catcgaaatt gccgtcaacc aagctctgat agagttggtc
9361 aagaccaatg cggagcatat acgcccggag ccgcggcgat cctgcaagct ccggatgcct
9421 ccgctcgaag tagcgcgtct gctgctccat acaagccaac cacggcctcc agaagaagat
9481 gttggcgacc tcgtattggg aatccccgaa catcgcctcg ctccagtcaa tgaccgctgt
9541 tatgcggcca ttgtccgtca ggacattgtt ggagccgaaa tccgcgtgca cgaggtgccg
9601 gacttcgggg cagtcctcgg cccaaagcat cagctcatcg agagcctgcg cgacggacgc
9661 actgacggtg tcgtccatca cagtttgcca gtgatacaca tggggatcag caatcgcgca
9721 tatgaaatca cgccatgtag tgtattgacc gattccttgc ggtccgaatg ggccgaaccc
9781 gctcgtctgg ctaagatcgg ccgcagcgat cgcatccatg gcctccgcga ccggctgcag
9841 aacagcgggc agttcggttt caggcaggtc ttgcaacgtg acaccctgtg cacggcggga
9901 gatgcaatag gtcaggctct cgctgaattc cccaatgtca agcacttccg gaatcgggag
9961 cgcggccgat gcaaagtgcc gataaacata acgatctttg tagaaaccat cggcgcagct
10021 atttacccgc aggacatatc cacgccctcc tacatcgaag ctgaaagcac gagattcttc
10081 gccctccgag agctgcatca ggtcggagac gctgtcgaac ttttcgatca gaaacttctc
10141 gacagacgtc gcggtgagtt caggcttttt catatcgggg tcgtcctctc caaatgaaat
10201 gaacttcctt atatagagga agggtcttgc gaaggatagt gggattgtgc gtcatccctt
10261 acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct
10321 ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca
10381 tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt
10441 tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc
10501 gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg
10561 atattcttgg agtagacgag agtgtcgtgc tccaccatgt tgacggatct ctaggacgcg
10621 tcctagaagc taattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
10681 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
10741 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgcc cgctcctttc
10801 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
10861 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
10921 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
10981 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
11041 atctcgggct attcttttga tttataaggg attttgccga tttcggaacc accatcaaac
11101 aggattttcg cctgctgggg caaaccagcg tggaccgctt gctgcaactc tctcagggcc
11161 aggcggtgaa gggcaatcag ctgttgcccg tctcactggt gaaaagaaaa accaccccag
11221 tacattaaaa acgtccgcaa tgtgttatta agttgtctaa gcgtcaattt gtttacacca
11281 caatatatcc tgccaccagc cagccaacag ctccccgacc ggcagctcgg cacaaaatca
11341 ccactcgata caggcagccc atcagtccgg gacggcgtca gcgggagagc cgttgtaagg
11401 cggcagactt tgctcatgtt accgatgcta ttcggaagaa cggcaactaa gctgccgggt
11461 ttgaaacacg gatgatctcg cggagggtag catgttgatt gtaacgatga cagagcgttg
11521 ctgcctgtga tcaaatatca tctccctcgc agagatccga attatcagcc ttcttattca
11581 tttctcgctt aaccgtgaca ggctgtcgat cttgagaact atgccgacat aataggaaat
11641 cgctggataa agccgctgag gaagctgagt ggcgctattt ctttagaagt gaacgttgac
11701 gatatcaact cccctatcca ttgctcaccg aatggtacag gtcggggacc cgaagttccg
11761 actgtcggcc tgatgcatcc ccggctgatc gaccccagat ctggggctga gaaagcccag
11821 taaggaaaca actgtaggtt cgagtcgcga gatcccccgg aaccaaagga agtaggttaa
11881 acccgctccg atcaggccga gccacgccag gccgagaaca ttggttcctg taggcatcgg
11941 gattggcgga tcaaacacta aagctactgg aacgagcaga agtcctccgg ccgccagttg
12001 ccaggcggta aaggtgagca gaggcacggg aggttgccac ttgcgggtca gcacggttcc
12061 gaacgccatg gaaaccgccc ccgccaggcc cgctgcgacg ccgacaggat ctagcgctgc
12121 gtttggtgtc aacaccaaca gcgccacgcc cgcagttccg caaatagccc ccaggaccgc
12181 catcaatcgt atcgggctac ctagcagagc ggcagagatg aacacgacca tcagcggctg
12241 cacagcgcct accgtcgccg cgaccccgcc cggcaggcgg tagaccgaaa taaacaacaa
12301 gctccagaat agcgaaatat taagtgcgcc gaggatgaag atgcgcatcc accagattcc
12361 cgttggaatc tgtcggacga tcatcacgag caataaaccc gccggcaacg cccgcagcag
12421 cataccggcg acccctcggc ctcgctgttc gggctccacg aaaacgccgg acagatgcgc
12481 cttgtgagcg tccttggggc cgtcctcctg tttgaagacc gacagcccaa tgatctcgcc
12541 gtcgatgtag gcgccgaatg ccacggcatc tcgcaaccgt tcagcgaacg cctccatggg
12601 ctttttctcc tcgtgctcgt aaacggaccc gaacatctct ggagctttct tcagggccga
12661 caatcggatc tcgcggaaat cctgcacgtc ggccgctcca agccgtcgaa tctgagcctt
12721 aatcacaatt gtcaatttta atcctctgtt tatcggcagt tcgtagagcg cgccgtgcgt
12781 cccgagcgat actgagcgaa gcaagtgcgt cgagcagtgc ccgcttgttc ctgaaatgcc
12841 agtaaagcgc tggctgctga acccccagcc ggaactgacc ccacaaggcc ctagcgtttg
12901 caatgcacca ggtcatcatt gacccaggcg tgttccacca ggccgctgcc tcgcaactct
12961 tcgcaggctt cgccgacctg ctcgcgccac ttcttcacgc gggtggaatc cgatccgcac
13021 atgaggcgga aggtttccag cttgagcggg tacggctccc ggtgcgagct gaaatagtcg
13081 aacatccgtc gggccgtcgg cgacagcttg cggtacttct cccatatgaa tttcgtgtag
13141 tggtcgccag caaacagcac gacgatttcc tcgtcgatca ggacctggca acgggacgtt
13201 ttcttgccac ggtccaggac gcggaagcgg tgcagcagcg acaccgattc caggtgccca
13261 acgcggtcgg acgtgaagcc catcgccgtc gcctgtaggc gcgacaggca ttcctcggcc
13321 ttcgtgtaat accggccatt gatcgaccag cccaggtcct ggcaaagctc gtagaacgtg
13381 aaggtgatcg gctcgccgat aggggtgcgc ttcgcgtact ccaacacctg ctgccacacc
13441 agttcgtcat cgtcggcccg cagctcgacg ccggtgtagg tgatcttcac gtccttgttg
13501 acgtggaaaa tgaccttgtt ttgcagcgcc tcgcgcggga ttttcttgtt gcgcgtggtg
13561 aacagggcag agcgggccgt gtcgtttggc atcgctcgca tcgtgtccgg ccacggcgca
13621 atatcgaaca aggaaagctg catttccttg atctgctgct tcgtgtgttt cagcaacgcg
13681 gcctgcttgg cctcgctgac ctgttttgcc aggtcctcgc cggcggtttt tcgcttcttg
13741 gtcgtcatag ttcctcgcgt gtcgatggtc atcgacttcg ccaaacctgc cgcctcctgt
13801 tcgagacgac gcgaacgctc cacggcggcc gatggcgcgg gcagggcagg gggagccagt
13861 tgcacgctgt cgcgctcgat cttggccgta gcttgctgga ccatcgagcc gacggactgg
13921 aaggtttcgc ggggcgcacg catgacggtg cggcttgcga tggtttcggc atcctcggcg
13981 gaaaaccccg cgtcgatcag ttcttgcctg tatgccttcc ggtcaaacgt ccgattcatt
14041 caccctcctt gcgggattgc cccgactcac gccggggcaa tgtgccctta ttcctgattt
14101 gacccgcctg gtgccttggt gtccagataa tccaccttat cggcaatgaa gtcggtcccg
14161 tagaccgtct ggccgtcctt ctcgtacttg gtattccgaa tcttgccctg cacgaatacc
14221 agcgacccct tgcccaaata cttgccgtgg gcctcggcct gagagccaaa acacttgatg
14281 cggaagaagt cggtgcgctc ctgcttgtcg ccggcatcgt tgcgccacat ctaggtacta
14341 aaacaattca tccagtaaaa tataatattt tattttctcc caatcaggct tgatccccag
14401 taagtcaaaa aatagctcga catactgttc ttccccgata tcctccctga tcgaccggac
14461 gcagaaggca atgtcatacc acttgtccgc cctgccgctt ctcccaagat caataaagcc
14521 acttactttg ccatctttca caaagatgtt gctgtctccc aggtcgccgt gggaaaagac
14581 aagttcctct tcgggctttt ccgtctttaa aaaatcatac agctcgcgcg gatctttaaa
14641 tggagtgtct tcttcccagt tttcgcaatc cacatcggcc agatcgttat tcagtaagta
14701 atccaattcg gctaagcggc tgtctaagct attcgtatag ggacaatccg atatgtcgat
14761 ggagtgaaag agcctgatgc actccgcata cagctcgata atcttttcag ggctttgttc
14821 atcttcatac tcttccgagc aaaggacgcc atcggcctca ctcatgagca gattgctcca
14881 gccatcatgc cgttcaaagt gcaggacctt tggaacaggc agctttcctt ccagccatag
14941 catcatgtcc ttttcccgtt ccacatcata ggtggtccct ttataccggc tgtccgtcat
15001 ttttaaatat aggttttcat tttctcccac cagcttatat accttagcag gagacattcc
15061 ttccgtatct tttacgcagc ggtatttttc gatcagtttt ttcaattccg gtgatattct
15121 cattttagcc atttattatt tccttcctct tttctacagt atttaaagat accccaagaa
15181 gctaattata acaagacgaa ctccaattca ctgttccttg cattctaaaa ccttaaatac
15241 cagaaaacag ctttttcaaa gttgttttca aagttggcgt ataacatagt atcgacggag
15301 ccgattttga aaccacaatt atgggtgatg ctgccaactt actgatttag tgtatgatgg
15361 tgtttttgag gtgctccagt ggcttctgtg tctatcagct gtccctcctg ttcagctact
15421 gacggggtgg tgcgtaacgg caaaagcacc gccggacatc agcgctatct ctgctctcac
15481 tgccgtaaaa catggcaact gcagttcact tacaccgctt ctcaacccgg tacgcaccag
15541 aaaatcattg atatggccat gaatggcgtt ggatgccggg caacagcccg cattatgggc
15601 gttggcctca acacgatttt acgtcactta aaaaactcag gccgcagtcg gtaacctcgc
15661 gcatacagcc gggcagtgac gtcatcgtct gcgcggaaat ggacgaacag tggggctatg
15721 tcggggctaa atcgcgccag cgctggctgt tttacgcgta tgacagtctc cggaagacgg
15781 ttgttgcgca cgtattcggt gaacgcacta tggcgacgct ggggcgtctt atgagcctgc
15841 tgtcaccctt tgacgtggtg atatggatga cggatggctg gccgctgtat gaatcccgcc
15901 tgaagggaaa gctgcacgta atcagcaagc gatatacgca gcgaattgag cggcataacc
15961 tgaatctgag gcagcacctg gcacggctgg gacggaagtc gctgtcgttc tcaaaatcgg
16021 tggagctgca tgacaaagtc atcgggcatt atctgaacat aaaacactat caataagttg
16081 gagtcattac ccaattatga tagaatttac aagctataag gttattgtcc tgggtttcaa
16141 gcattagtcc atgcaagttt ttatgctttg cccattctat agatatattg ataagcgcgc
16201 tgcctatgcc ttgccccctg aaatccttac atacggcgat atcttctata taaaagatat
16261 attatcttat cagtattgtc aatatattca aggcaatctg cctcctcatc ctcttcatcc
16321 tcttcgtctt ggtagctttt taaatatggc gcttcataga gtaattctgt aaaggtccaa
16381 ttctcgtttt catacctcgg tataatctta cctatcacct caaatggttc gctgggttta
16441 tcgcaccccc gaacacgagc acggcacccg cgaccactat gccaagaatg cccaaggtaa
16501 aaattgccgg ccccgccatg aagtccgtga atgccccgac ggccgaagtg aagggcaggc
16561 cgccacccag gccgccgccc tcactgcccg gcacctggtc gctgaatgtc gatgccagca
16621 cctgcggcac gtcaatgctt ccgggcgtcg cgctcgggct gatcgcccat cccgttactg
16681 ccccgatccc ggcaatggca aggactgcca gcgctgccat ttttggggtg aggccgttcg
16741 cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgggc cgggagggtt
16801 cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg cgcacagggc gcagccctgg
16861 ttaaaaacaa ggtttataaa tattggttta aaagcaggtt aaaagacagg ttagcggtgg
16921 ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc tgcctgtgga cagcccctca
16981 aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc cctcaagtgt caaggatcgc
17041 gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat accgcagggc acttatcccc
17101 aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc aggcgttttc gccgatttgc
17161 gaggctggcc agctccacgt cgccggccga aatcgagcct gcccctcatc tgtcaacgcc
17221 gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc ctcatctgtc agtgagggcc
17281 aagttttccg cgaggtatcc acaacgccgg cggccgcggt gtctcgcaca cggcttcgac
17341 ggcgtttctg gcgcgtttgc agggccatag acggccgcca gcccagcggc gagggcaacc
17401 agcccgg
//
质粒菌株产品操作说明书
一、扩增流程
收到产品后，请先根据产品管壁标签来判断产品形式，并在扩增前准确查找该质粒菌株的抗性、感受态和培养温度。
1、质粒干粉（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
①收到质粒干粉后请先5000rpm离心1min，再加入20μl ddH2O去离子水溶解质粒；
②取1支100μl 感受态于冰上解冻10min，加入2μl质粒，再冰浴30min后，42℃热激60s，不要搅动，再冰浴2min；（从第二步开始均要在超净工作台中无菌操作）
③加入900μl无抗的LB液体培养基，180rpm震荡37℃培养45min；
④6000rpm离心5min，仅留100μl上清液重悬细菌沉淀，并涂布至目标质粒抗性的LB平板上；（可使用本平台的平板涂布专用玻璃珠进行涂布，可以比传统涂布方法获得更多转化子）
⑤将平板正向培养1h，再倒置37℃培养14h。如果要求是30度则培养20h；
（菌落过多则将质粒稀释后再转化。没有菌落则加入10μl质粒转化。另不要直接转表达感受态，要先转克隆感受态，重提质粒后再导入表达感受态）
⑥挑取单菌落至LB液体培养基中，加入对应抗生素，220rpm震荡培养14h，根据实验需要和质粒提取试剂盒说明书提取质粒。
2、甘油菌种（冰袋运输，存于-80℃，保质期90天，请务必划线挑单克隆培养）
四区划线后挑单菌落培养，酵母菌需要先液体复苏再四区划线，再挑单菌落液体培养。
3、穿刺菌种（冰袋运输，存于4℃，保质期7天）
穿刺接种，液体培养后四区划线，再挑单菌落液体培养。
4、菌落平板（冰袋运输，存于4℃，保质期7天）
直接挑取单菌落至液体培养基中。
5、液体质粒（冰袋运输，存于-20℃，保质期90天）
单独提取的液体质粒收到后可直接使用。
6、滤纸质粒（常温运输，存于-20度，90天保质期，请务必转化挑单克隆培养，不要直接使用和测序）
收到货后将滤纸画圈部分剪下放入EP管中，加100ul无菌水将滤纸浸湿并浸泡5min，吸取5ul质粒转化，离心全涂。
二、转化图片
| P2817/pENTR223-MLC1-C74A人源基因质粒 |
| P2818/pENTR223-GNAZ-C404A人源基因质粒 |
| P2819/pENTR223-FGD6人源基因质粒 |
| P2820/pENTR223-IQUB（1-1119bp）人源基因质粒 |
| P2821/pENTR223-PTPN2-C15G人源基因质粒 |
| P2822/pENTR223-NFKBIE（418-1503bp）人源基因质粒 |
| P2823/pENTR223-DNAJB11人源基因质粒 |
| P2824/pENTR223-B4GALT3（2同义突变）人源基因质粒 |
| P2825/pENTR223-KCNJ13（3点突变）人源基因质粒 |
| P2848/pENTR223-NGEF人源基因质粒 |
| P1600/pENTR-GFP-MDC1 (770-7)人源基因质粒 |
| P1337/R777-E011 Hs.ARHGAP35人源基因质粒 |
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pGWB17植物表达质粒
￥100 - 1000
